plasmid cloning - i need help.. can anyone answer me (Dec/02/2010 )

hi .. i was wondering about plasmid cloning and exactly why some cells (E.coli bacteria for example ) take up plasmid and the others don't

-ntdna-

Why are some plasmids able to enter cells (ex: EcoRI)? What unique properties do they possess?

-ntdna-

It depends on the conditions...

EcoR1 is not a plasmid.

-bob1-

bob1 on Mon Dec 6 22:48:43 2010 said:

It depends on the conditions...

EcoR1 is not a plasmid.

i know ecor1 is not a plamid it's a restrction enzyme

i said Why are some plasmids able to enter cells (ex: Ecoli )? What unique properties do they possess?in plasmid cloning

-ntdna-

ntdna on Tue Dec 7 00:34:49 2010 said:

bob1 on Mon Dec 6 22:48:43 2010 said:

EcoR1 is not a plasmid.

i know ecor1 is not a plamid it's a restrction enzyme

They why did you call it one in your second post?

There's nothing unique about E. coli, you can easily transform many other species, E. coli is just abundant so easily obtainable.

Try reading Hanahan 1989 for reasons he used it.

Note - I'm being obtuse because this really really appears to be homework, so you should be thinking about potential reasons yourself.

-bob1-

The ability to take up exogenous DNA is an evolutionary adaptation. Think about it in these terms -- what evolutionary advantage(s) might be gained by a species that can take up and incorporate exogenous DNA? Protection against the introduction of foreign DNA into the cell is also an evolutionary adaptation (e.g. restriction enzymes and methylases). How are such systems advantageous (think viruses...)?

-HomeBrew-

BL21 would be a very bad choice of strain to attempt cloning with. Your choices boil down to finding and using a more stable cloning strain, or finding out more about what exactly is happening. Do you have sequence for the construct you want? Do you have sequence for the plasmid that you created? That would be my first order of business. Then, I would look at what repeats and homologies exist that might explain the recombination you are apparently seeing. You'll know a lot more after doing this, if only to know that you have a severe problem. More likely, you will discover the problem is something entirely else that we haven't thought of. You should be collecting all of the information you can, using whatever tools are available, to figure out what is happening. Have you done restriction digests of the plasmids you create? These are simple and inexpensive. You can't figure out what is wrong by asking others or by waiting.